Novel Prion Strain as Cause of Chronic Wasting Disease in a Moose, Finland

Our previous studies using gene-targeted mouse models of chronic wasting disease (CWD) demonstrated that Norway and North America cervids are infected with distinct prion strains that respond differently to naturally occurring amino acid variation at residue 226 of the prion protein. Here we performed transmissions in gene-targeted mice to investigate the properties of prions causing newly emergent CWD in moose in Finland. Although CWD prions from Finland and Norway moose had comparable responses to primary structural differences at residue 226, other distinctive criteria, including transmission kinetics, patterns of neuronal degeneration, and conformational features of prions generated in the brains of diseased mice, demonstrated that the strain properties of Finland moose CWD prions are different from those previously characterized in Norway CWD. Our findings add to a growing body of evidence for a diverse portfolio of emergent strains in Nordic countries that are etiologically distinct from the comparatively consistent strain profile of North America CWD.

Although they lack nucleic acids, prions exhibit heritable strain properties that influence disease outcomes, including the time between infection and disease onset (incubation period), clinical signs, patterns of neuronal degeneration and PrP Sc deposition in the central nervous system (CNS), and the ability to replicate in non-CNS tissues such as the lymphoreticular system and musculature (12). Strain properties also influence the capacity of prions from one species to cause disease in a different species (13). Heritable strain information appears to be enciphered by distinct PrP Sc conformations that are faithfully propagated during prion replication (14,15).
The food-chain transmission of bovine spongiform encephalopathy prions that resulted in a variant form of CJD illustrates the unpredictable potential of emergent strains for zoonotic transmission (13). Although novel prion diseases and strain variants continue to arise in increasing numbers of animal species, CWD elicits particular concern (16). After its initial description in a captive deer facility (17), uncontrolled contagious transmission has resulted in growing numbers of CWD-affected cervids in at least 30 US states and 3 Canada provinces (18). Inadvertent importation of subclinically diseased animals from North America led to the establishment of CWD in South Korea (19)(20)(21). CWD was also diagnosed in free-ranging Norwegian reindeer in 2016 (22), and additional cases were subsequently identified in growing numbers of moose, red deer, and reindeer from Norway, Sweden, and Finland (23).
The development and application of genetically modified, CWD-susceptible mice expressing cervid PrP C (CerPrP C ) has provided insights into multiple

Novel Prion Strain as Cause of Chronic Wasting Disease in a Moose, Finland
Our previous studies using gene-targeted mouse models of chronic wasting disease (CWD) demonstrated that Norway and North America cervids are infected with distinct prion strains that respond differently to naturally occurring amino acid variation at residue 226 of the prion protein.
Here we performed transmissions in gene-targeted mice to investigate the properties of prions causing newly emergent CWD in moose in Finland. Although CWD prions from Finland and Norway moose had comparable responses to primary structural differences at residue 226, other distinctive criteria, including transmission kinetics, patterns of neuronal degeneration, and conformational features of prions generated in the brains of diseased mice, demonstrated that the strain properties of Finland moose CWD prions are different from those previously characterized in Norway CWD. Our findings add to a growing body of evidence for a diverse portfolio of emergent strains in Nordic countries that are etiologically distinct from the comparatively consistent strain profile of North America CWD.
aspects of pathogenesis, including the impact of naturally occurring PRNP coding sequence variations (16). Whereas North America deer and moose encode glutamine (Q) at codon 226 (CerPrP-Q226), North America elk encode glutamate (E) at this position (CerPrP-E226). Early studies in transgenic (Tg) mice suggested a role for this variation in the selection and propagation of CWD strains (24)(25)(26). To precisely assess the effects of this primary structural difference, we created CWDsusceptible gene-targeted (Gt) mice in which the PrP coding sequence was replaced with CerPrP-Q226 or CerPrP-E226 (27). Because the resulting mice, referred to as GtQ and GtE mice, express equivalent, physiologically controlled levels of CerPrP C and are otherwise syngeneic, we were able to ascribe distinct disease outcomes in each line to the effects of these amino acids (27). Furthermore, in contrast to previous CWD-susceptible Tg mice, Gt mice recapitulated the lymphotropic properties of CWD strains (27,28). By using Gt mice, we showed that emergent strains causing CWD in Norway reindeer and moose were unrelated to established North American forms of CWD and that they responded differently to variation at residue 226 (28). These findings underscored the utility of Gt mice for accurately defining the strain properties of emergent CWD prions (28). The goal of this study was to characterize a newly emergent form of CWD in Finland moose.

Ethics Statement
We performed all animal work in an Association for Assessment for Accreditation of Laboratory Animal Careaccredited facility in accordance with the Guide for the Care and Use of Laboratory Animals (https://www. ncbi.nlm.nih.gov/books/NBK54050). All procedures used in this study were performed in compliance with and were approved by the Colorado State University Institutional Animal Care and Use Committee.

Isolates
The Finland moose (M-F1) CWD isolate was from a free-ranging female moose identified to be sick and later found dead in 2018. The North America moose (M-US1) CWD isolate was from a captive moose orally inoculated with a pooled preparation of deer CWD. North America elk CWD isolate (E-US1), and Norway moose CWD isolates (M-NO1, M-NO2, and M-NO3) have been described previously (28).

Mouse Models and Incubation Time Assay
Development and characterization of Tg mice expressing cervid PrP with glutamine at residue 226 (TgQ), Tg mice expressing cervid PrP with glutamate at residue 226 (TgE), GtQ and GtE mice (expressing the same as the Tg mice), and the prion incubation time assay have been described previously (24,(27)(28)(29). We used equal numbers of male and female mice in inoculation studies. We confirmed clinical diagnoses of prion disease by PrP Sc detection in the CNS and neuropathologic assessments.

Conformational Stability Assay
We conducted a conformational stability assay as described previously (27,28). We detected PrP Sc on membranes with mAb PRC5 (30) at a dilution of 1:5,000, followed by horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody. We developed membranes with ECL 2 Western Blotting Substrate and scanned them with an ImageQuant LAS 4000 digital camera system (GE Healthcare, https://www. gehealthcare.com) and analyzed signals with Im-ageQuant TL 7.0 software (GE Healthcare).
We dissected brains rapidly after euthanizing the animal and immersion fixed tissues in 10% buffered formalin. We embedded tissues in paraffin and mounted 8 μm-thick coronal microtome sections onto positively charged glass slides. We performed immunohistochemical analyses of PrP in brain sections by using mAb D18 as described previously (28).

Neuropathologic Lesion Profiling
We sectioned paraffin-embedded mouse brains coronally to areas corresponding to 5 levels of the brain containing the 9 mouse brain regions of interest. We captured images of hematoxylin and eosin-stained sections by using a Photometrics CoolSNAP digital camera (https://www.photometrics.com) and an Olympus BX51 fluorescence microscope (https:// www.olympus-lifescience.com) and analyzed them by using Olympus cellSens imaging software (standard version). We assessed the severity of vacuolar degeneration in cerebral gray matter from 9 regions, as described previously (32), by using a team of 4 investigators, who scored the images on scale of 0 to 4, with 4 corresponding to severe vacuolation.

Statistical Analyses
We performed statistical analyses by using Graph-Pad Prism 8.0 software (https://www.graphpad. com). We assessed statistical significance between survival curves of inoculated groups by comparing median times of survival of various inoculated groups using the log-rank (Mantel-Cox) test. We assessed statistical significance between denaturation curves of PrP Sc by comparing log half maximal effective concentration values.

Results
We registered disease after ≈260 days in all but 1 of 11 GtQ mice inoculated with a homogenate of CNS from the index case of Finland CWD, M-F1 (Table 1; Figure 1, panel A). Brain homogenates from 2 diseased GtQ mice, referred to as GtQ (M-F1) brain 1 and brain 2 ( Figure 1, panel A), produced disease after ≈225 days following intracerebral inoculation of additional GtQ mice (Table 2; Figure 1, panel D, E). By contrast, all GtE mice inoculated with M-F1 brain homogenate remained free of disease for up to 545 days (Table 1; Figure 1, panel A), and GtQ (M-F1) brain 1 produced disease in only 4 of 7 inoculated GtE mice after ≈470 days (Table 2; Figure 1, panel D), whereas GtQ (MF-1) brain 2 failed to produced disease in GtE mice after >510 days (Table 2; Figure 1, panel E). We conclude that M-F1 prion replication is supported when Q is encoded at residue 226 of hostencoded PrP (CerPrP C -Q226) and relatively restricted when E is present at that position. In support of this conclusion, whereas CNS overexpression of CerPrP C -Q226 produced faster times to disease in TgQ compared with GtQ mice, only a single inoculated TgE mouse had disease after ≈390 days (Appendix Table  1, Figure 1, panel A, https://wwwnc.cdc.gov/EID/ article/29/2/22-0882-App1.pdf).
We confirmed clinical diagnoses of prion disease by using western blotting of PK-resistant CerPrP Sc in brain homogenates ( disease in GtE mice after ≈500 days (Appendix Table 2, Figure 1, panel E). Because serial passage of M-F1 prions from diseased TgQ mice also produced disease in GtQ mice after ≈230 days and failed to produce disease in GtE mice after ≈500 days (Appendix Table 2, Figure  1, panel D), we conclude that the M-F1 strain favors conformational conversion of CerPrP C -Q226 over Cer-PrP C -E226 regardless of whether prions are composed of CerPrP Sc -E226 or CerPrP Sc -Q226.
Our previous studies showed that propagation of prions from 3 Norway moose CWD isolates, referred to as M-NO1, M-NO2, and M-NO3, was also supported by expression of CerPrP C -Q226 and relatively restricted by CerPrP C -E226 (28). Despite this shared feature, comparisons of primary transmissions to GtQ mice showed that the mean incubation time of M-F1 was faster than those of M-NO1, M-NO2, or M-NO3 (p<0.0001) ( Table  1; Figure 1, panel C). Primary transmission of M-F1 was also faster than Norway moose CWD isolates in TgQ mice (p>0.0001) (Appendix Table 1, Figure 1, panel C). We also observed faster disease onsets when serial transmissions of GtQ (M-F1) brains 1 and 2 were compared with serially transmitted M-NO1 and M-NO2 in GtQ mice (Table 2; Figure 1, panel F) and when M-F1 and Norway moose CWD prions were serially propagated from TgQ to GtQ mice (Appendix Table 2, Figure  1, panel F). Those consistently different disease kinetics during primary and secondary transmissions are indicative of strain differences between Finland and Norway moose CWD prions.
To substantiate that conclusion, we assessed additional strain features in infected GtQ mice. Semiquantitative assessment by brain lesion profiling (32)     showed that the extent and regional distribution of spongiform degeneration varied depending on whether GtQ mice were infected with Finland or Norway moose CWD prions (Figure 4). Differences between GtQ mice infected with M-F1 and M-NO1 were particularly evident in the septum, retrosplenial and adjacent motor cortex, and cingulate and adjacent motor cortex. In these areas the intensity of spongiform degeneration was more pronounced in GtQ mice infected with M-F1 (Figure 4; Appendix Figure 3, panel A, B). The distinctive lesion profile of M-F1 was sustained after serial passage to additional GtQ mice (Figure 4).
We assessed the responses of CerPrP Sc produced in the brains of mice infected with Finland, Norway, and North America moose CWD prions to denaturation with increasing concentrations of guanidine hydrochloride (GdnHCl) (Figure 6). This measure of PrP Sc stability is associated with conformational variation among prion strains (33). Denaturation profiles and concentrations of GdnHCl producing half-maximal denaturation indicated that the stability of CerPrP Sc in GtQ mice infected with M-F1 was equivalent to CerPrP Sc in the brains of GtQ mice infected with North America moose CWD and lower than that of CerPrP Sc in the brains of GtQ mice infected with M-NO1 (p<0.0001) (Figure 6, panel A). The conformational stability of CerPrP Sc produced on primary passage of M-F1 was maintained upon serial transmissions of GtQ (M-F1) brains 1 and 2 to GtQ mice ( Figure 6, panel B). We also observed overlapping denaturation curves and comparable half-  M-NO1 prions, they are stable during iterative passages, and they remain unchanged after passage in mice expressing CerPrP C -E226.
Despite their different transmission properties, neuropathologic profiles, and conformations, Finland and Norway CWD prions shared certain features. Western blotting showed that levels of CerPrP Sc in the brains of GtQ mice infected with M-F1 and M-NO1 were lower than the levels of CerPrP Sc in the brains of GtQ mice infected with North America CWD ( Figure  2). This finding is consistent with previous studies showing consistently reduced CerPrP Sc accumulation after Norway moose CWD prion infection (28). The electrophoretic migration profiles of CerPrP Sc resulting from infection of GtQ mice with M-F1 and M-NO1 were both more rapid than CerPrP Sc produced by infection with North America CWD (Figure 2, panel A). This finding also is consistent with our previous descriptions of Norway moose CerPrP Sc (28). Finally, also in accordance with previous findings (28), CerPrP Sc in the brains of M-F1 and M-NO1 infected mice was relatively refractory to detection by mAb PRC1 compared with CerPrP Sc generated by infection with North America CWD (Figure 2, panel B). The faster migration of CerPrP Sc generated by infection with M-F1 and M-NO1 and its comparative resistance to detection by PRC1 results from PK cleavage downstream from the PRC1 epitope at residue 90 in both strains (Appendix Figure 2). Serial transmissions of M-F1, M-NO1, and North America CWD  prions produced CerPrP Sc immunoblotting profiles consistent with primary passages (Figure 2, panel C, D). We speculate that the low levels of PRC1-reactive CerPrP Sc in brain extracts of mice infected with M-F1 and M-NO1 CWD prions (Figure 2, panel B, D) correspond to minor strain components that remain obscured when probing with nondiscriminatory mAbs.
Using discriminatory mAbs revealed similar coexistence of multiple PrP Sc types in persons with CJD (34). Analyses of PK-resistant CerPrP Sc in histoblotted coronal brain sections revealed a diffuse, symmetric deposition pattern in GtQ mice infected with M-F1 (Figure 3, panel A) that was indistinguishable from the pattern in GtQ mice infected with M-NO1 CWD (Figure 3, panel D), and consistent with previous analyses of mice infected with Norway moose CWD (28). This pattern was recapitulated after iterative passage of GtQ (M-F1) prions to GtQ mice (

Discussion
Our previous studies showed that although Gt and Tg mice expressing either CerPrP C -E226 or CerPrP C -Q226 were susceptible to North America CWD, times to disease onset were more rapid in mice expressing CerPrP C -E226 (24,(26)(27)(28). By comparison, although efficient transmission of Norway moose CWD prions was registered in Gt and Tg mice expressing CerPrP C -Q226, their counterparts expressing CerPrP C -E226 were resistant to infection or had prolonged incubation times and incomplete attack rates (28). Those studies also revealed strain variation among Norway moose CWD isolates, the properties of which were different from Norway reindeer CWD (28). The different properties of emergent Norway strains compared with North America CWD made it unlikely that they were causally related. Parallel experiments in bank voles supported this interpretation (35). Differences in the strain properties of M-F1 and North America CWD enable us to build on our previous conclusions (28,35) and to state more broadly that the etiology of Nordic CWD is distinct from North America CWD.
Our analyses reveal that certain characteristics of M-F1 CWD prions overlap with those of Norway moose CWD. M-F1 and Norway moose CWD both propagated more efficiently in mice expressing Cer-PrP C -Q226. Western and histoblot profiles of M-F1 and M-NO1 CerPrP Sc are also indistinguishable. Our findings in this study show that M-F1 prions, like Norway moose CWD prions, are nonlymphotropic. Despite these broadly similar characteristics, other measures, including consistently different disease kinetics, distinct neuropathologic lesion profiles, and variable responses to GdnHCl denaturation, indicate that the strain properties of Finland and Norway moose CWD prions are different. This finding adds to an increasing body of evidence for a surprising variety of strains among Nordic cervids, which stands in contrast to the relatively consistent CWD strain profile among North America deer, elk, and moose. As yet, there appears to be no clear explanation for this diversity of Nordic CWD strains or insights into their origins. Although efficient prion dissemination to peripheral tissues of affected cervids has been cited as a reason for the contagious properties of North America CWD (36), the nonlymphotropic properties of Finland and Norway moose CWD strains suggests a decreased potential for contagious propagation. The emergence of increasing numbers of CWD strains in a confined geographic location during a short timeframe presents challenges when considering a sporadic etiology; however, our findings showing dissimilar CWD profiles in Nordic moose can be explained by divergent strains arising from unrelated, stochastic conversion events in different animals. CWD has also been diagnosed in moose in Sweden (23). Analyses of the strain properties of those additional emergent CWD cases will be of considerable interest.
Additional findings from this study build on our previous observations and warrant discussion.  Seminal studies indicating that related PrP Sc and PrP C primary structures supported optimal prion propagation laid the foundation for the development of Tg mice with susceptibility to human and animal prions (37)(38)(39). Because moose express CerPrP C -Q226, we reasoned that the relatively inefficient transmission prions to adapt to a host expressing a single amino acid difference is reminiscent of our description of nonadaptive prion amplification (NAPA) (40). Those studies showed that prions produced during particular interspecies transmissions are not adapted for sustained propagation in that new host. Instead, prions resulting from NAPA retain atavistic preferences for their species of origin (40). In this context, although M-F1 prions are capable of suboptimal propagation by NAPA using CerPrP C -E226 as template for replication, the resulting TgE (M-F1) prions are not adapted for further propagation by CerPrP C -E226 but instead retain a preference for conversion of CerPrP C -Q226.